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Olga Riccioni,1,2 Charalambos Vrasidas,3 Luka Brcic,4 Goce Armenski,5 Sven Seiwerth,4 Annemieke Smeets,6 J Han JM van Krieken,6 Andreas C Lazaris1 11st Department of Pathology, School of Medicine, The National and Kapodistrian University of Athens, Athens, Greece; 2School of Medicine, La Sapienza University of Rome, Rome, Italy; 3Centre for the Advancement of Research and Development in Educational Technology LTD, Nicosia, Cyprus; 4Institute of Pathology, School of Medicine, University of Zagreb, Zagreb, Croatia; 5Faculty of Computer Science and Engineering, Saints Cyril and Methodius University, Skopje, FYROM/Republic of Macedonia; 6Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Abstract: It is indisputable that nowadays one of the hardest and most important tasks in medicine and especially in medical education, is the conversion of the extensive amount of available data, into medical experience, after a proper analysis. A project under the title "ICT (Information and Communication Technology) eModules on HistoPathology: a useful online tool for students, researchers and professionals – HIPON", co-financed by the Lifelong Learning Program of the Education, Audiovisual and Culture Executive Agency (EACEA), The Commission of the European Union, has been launched at the beginning of 2013. HIPON's purpose is not to provide just another pathology website atlas, but to convey professional experience and thinking in pathology. HIPON has resulted in a well-structured and user-friendly, open resource, multi-language, e-learning platform which, taking advantage of modern image technology, offers medical students, researchers, and professionals a valuable teaching instrument so that they can acquire professional experience in pathology. The mid-term report of HIPON has been favorably evaluated by the EACEA experts who appreciated the potential of our teaching tool in providing the opportunity and the means to acquire medical experience. Through the use of virtual slides, educative videos and microscopic, high resolution, marked images accompanied by relevant questions and answers, HIPON project aims to make end-users able to think as experienced pathologists and become highly efficient in correlating pathologic data with other clinical-laboratory information. Keywords: international medical education, website, educational technology, case study

Background. Interleukin (IL)-1 alpha and IL-1 beta are protective proinflammatory cytokines involved in host defense against Candida albicans. It is, however, unknown whether they provide protection through similar mechanisms. We investigated the effect of endogenous IL-1 alpha and IL-1 beta on disseminated C. albicans infection. Methods. Mice deficient in the genes encoding IL-1 alpha (IL-1 alpha (-/-)), IL-1 beta (IL-1 beta (-/-)), or both molecules (IL-1 alpha (-/-) beta (-/-)) were used. Survival and C. albicans outgrowth in the kidneys was assessed after intravenous injection of C. albicans.Results. Both mortality and C. albicans outgrowth in the kidneys were significantly increased in IL-1 alpha (-/-) and IL-1 beta (-/-) mice, compared with those in control mice, with the IL-1 alpha (-/-) beta (-/-) mice being most susceptible to disseminated candidiasis. The host defense mechanisms triggered by IL-1 alpha and IL-1 beta differed from one another. IL-1 beta (-/-) mice showed decreased recruitment of granulocytes in response to an intraperitoneal C. albicans challenge, and generation of superoxide production was diminished in IL-1 beta (-/-) granulocytes. IL-1 alpha (-/-) mice had a reduced capacity to damage C. albicans pseudohyphae. Protective type 1 responses were deficient in both IL-1 alpha (-/-) and IL-1 beta (-/-) mice, as assessed by production of interferon- gamma by splenocytes in response to heat-killed C. albicans.Conclusion. Although IL-1 alpha and IL-1 beta have differential effects on the various arms of host defense, both cytokines are essential for mounting a protective host response against invasive C. albicans infection. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Purpose: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance., Experimental Design: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution., Results: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell-like/activated B-cell-like classification and TIL-T frequency. The identified TIL-B-high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts., Conclusions: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications., (©2023 American Association for Cancer Research.)

Purpose: Diffuse large B-cell lymphoma (DLBCL) is molecularly and clinically heterogeneous, and can be subtyped according to genetic alterations, cell-of-origin, or microenvironmental signatures using high-throughput genomic data at the DNA or RNA level. Although high-throughput proteomic profiling has not been available for DLBCL subtyping, MYC/BCL2 protein double expression (DE) is an established prognostic biomarker in DLBCL. The purpose of this study is to reveal the relative prognostic roles of DLBCL genetic, phenotypic, and microenvironmental biomarkers., Experimental Design: We performed targeted next-generation sequencing; IHC for MYC, BCL2, and FN1; and fluorescent multiplex IHC for microenvironmental markers in a large cohort of DLBCL. We performed correlative and prognostic analyses within and across DLBCL genetic subtypes and MYC/BCL2 double expressors., Results: We found that MYC/BCL2 double-high-expression (DhE) had significant adverse prognostic impact within the EZB genetic subtype and LymphGen-unclassified DLBCL cases but not within MCD and ST2 genetic subtypes. Conversely, KMT2D mutations significantly stratified DhE but not non-DhE DLBCL. T-cell infiltration showed favorable prognostic effects within BN2, MCD, and DhE but unfavorable effects within ST2 and LymphGen-unclassified cases. FN1 and PD-1-high expression had significant adverse prognostic effects within multiple DLBCL genetic/phenotypic subgroups. The prognostic effects of DhE and immune biomarkers within DLBCL genetic subtypes were independent although DhE and high Ki-67 were significantly associated with lower T-cell infiltration in LymphGen-unclassified cases., Conclusions: Together, these results demonstrated independent and additive prognostic effects of phenotypic MYC/BCL2 and microenvironment biomarkers and genetic subtyping in DLBCL prognostication, important for improving DLBCL classification and identifying prognostic determinants and therapeutic targets., (©2022 American Association for Cancer Research.)

Multiple studies have demonstrated that diffuse large B-cell lymphoma (DLBCL) can be divided into subgroups based on their biology; however, these biological subgroups overlap clinically. Using machine learning, we developed an approach to stratify patients with DLBCL into four subgroups based on survival characteristics. This approach uses data from the targeted transcriptome to predict these survival subgroups. Using the expression levels of 180 genes, our model reliably predicted the four survival subgroups and was validated using independent groups of patients. Multivariate analysis showed that this patient stratification strategy encompasses various biological characteristics of DLBCL, and only TP53 mutations remained an independent prognostic biomarker. This novel approach for stratifying patients with DLBCL, based on the clinical outcome of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone therapy, can be used to identify patients who may not respond well to these types of therapy, but would otherwise benefit from alternative therapy and clinical trials., (© 2022. The Author(s).)

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma with high mutation burdens but a low response rate to immune checkpoint inhibitors. In this study, we performed targeted next-generation sequencing and fluorescent multiplex immunohistochemistry, and investigated the clinical significance and immunological effect of mutation numbers in 424 DLBCL patients treated with standard immunochemotherapy. We found that KMT2D and TP53 nonsynonymous mutations (MUT) were significantly associated with increased nonsynonymous mutation numbers, and that high mutation numbers (MUT high ) were associated with significantly poorer clinical outcome in germinal center B-cell-like DLBCL with wild-type TP53 . To understand the underlying mechanisms, we identified a gene-expression profiling signature and the association of MUT high with decreased T cells in DLBCL patients with wild-type TP53 . On the other hand, in overall cohort, MUT high was associated with lower PD-1 expression in T cells and PD-L1 expression in macrophages, suggesting a positive role of MUT high in immune responses. Analysis in a whole-exome sequencing dataset of 304 patients deposited by Chapuy et al. validated the correlation of MUT- KMT2D with genomic complexity and the significantly poorer survival associated with higher numbers of genomic single nucleotide variants in activated B-cell-like DLBCL with wild-type TP53 . Together, these results suggest that KMT2D inactivation or epigenetic dysregulation has a role in driving DLBCL genomic instability, and that genomic complexity has adverse impact on clinical outcome in DLBCL patients with wild-type TP53 treated with standard immunochemotherapy. The oncoimmune data in this study have important implications for biomarker and therapeutic studies in DLBCL., Competing Interests: HN, MLN, and QA are employees of NeoGenomics Laboratories, Inc.. MA is an employee of Genomic Testing Corporative, LCA. Other authors declare no conflicts of interest., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: The literature on biomarker testing for metastatic colorectal cancer (mCRC) in Europe is scarce. This study aimed to estimate the percentage of mCRC patients from five European countries tested for biomarkers over time. Materials & methods: An oncology database was retrospectively analyzed; evaluated biomarkers were RAS , BRAF  and microsatellite instability (MSI). The patients were drug treated during 2018 and tested for relevant biomarkers in 2013-2018. Results: RAS testing was conducted in >90% of mCRC patients from 2014 onwards. BRAF testing increased from 31% of mCRC patients in 2013 to 67% in 2018. MSI testing increased from 10 to 41%. There was no notable trend over time for RAS and BRAF mutation or MSI-high prevalence. Conclusion: Biomarker testing among patients diagnosed with mCRC was increased over time. This study demonstrates the quick uptake of biomarker testing in clinical practice. These findings are significant as biomarker-based drugs are becoming more common.

Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC / BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities ( MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2-targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL- MYC / BCL2 -DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC / BCL2 / TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. IMPLICATIONS: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg., (©2020 American Association for Cancer Research.)

The majority of patients with diffuse large B-cell lymphoma (DLBCL) can be treated successfully with a combination of chemotherapy and the monoclonal anti-CD20 antibody rituximab. Nonetheless, approximately one-third of the patients with DLBCL still experience relapse or refractory (R/R) disease after first-line immunochemotherapy. Whole-exome sequencing on large cohorts of primary DLBCL has revealed the mutational landscape of DLBCL, which has provided a framework to define novel prognostic subtypes in DLBCL. Several studies have investigated the genetic alterations specifically associated with R/R DLBCL, thereby uncovering molecular pathways linked to therapy resistance. Here, we summarize the current state of knowledge regarding the genetic alterations that are enriched in R/R DLBCL, and the corresponding pathways affected by these gene mutations. Furthermore, we elaborate on their potential role in mediating therapy resistance, also in connection with findings in other B-cell malignancies, and discuss alternative treatment options. Hence, this review provides a comprehensive overview on the gene lesions and molecular mechanisms underlying R/R DLBCL, which are considered valuable parameters to guide treatment.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell-like compared with the germinal-center B-cell-like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL., (© 2020 by The American Society of Hematology.)

Background: Diffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma., Methods: To determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens' immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry., Results: SHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHM high ) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell-like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHM high groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8 + T cells was significantly lower in IGHV SHM high than in SHM low patients with activated B-cell-like DLBCL, whereas PD-1 expression in CD4 + T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHM high than in SHM low patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM., Conclusions: These results show for the first time that IGV SHM high and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37 -knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations., (© 2019 by The American Society of Hematology.)

Background: Colon cancer treatment is dependent on the stage at diagnosis. The current Tumor-Node-Metastasis (TNM) staging for the selection of patients for adjuvant chemotherapy needs additional prognostic and predictive biomarkers. Better decision making for chemotherapy will result in reducing over- and undertreatment. We developed a new, easy-to-apply, practice-changing method to select colon cancer patients for adjuvant chemotherapy: the tumor-stroma ratio (TSR). The TSR distinguishes within stage II-III patients who will likely benefit from adjuvant chemotherapy and those who will not., Objective: The aim of the study was to add, in addition to the TNM classification, the TSR to current routine pathology evaluation. Pathologists will be instructed for scoring the TSR in combination with a quality assessment program. An international multicenter study will validate the parameter prospectively., Methods: The study is designed for future implementation of the TSR to the current TNM guidelines, using routinely Haematoxylin- and Eosin-stained tumor tissue sections. In part 1 of the study, an electronic learning (e-learning) module with a quality assessment program using the European Society of Pathology framework will be developed. This module will be used to assess the reliability and reproducibility of the TSR, conducted by national and international pathologists. Part 2 will involve the validation of the TSR in a prospective cohort of colon cancer p-stage II-III patients in a multicenter setting. In total, 1500 patients will be included., Results: The results of part 1 will be expected in the first half of 2019. For part 2, the inclusion of patients in the prospective study, which started at the end of 2018, will take 3 years with an additional follow-up after another 3 years., Conclusions: The main endpoints of this study are as follows: in part 1, trained (international) pathologists who are able to reliably score the TSR, resulting in low intra- and interobserver variation; in part 2, confirmation of significant survival differences for patients with a stroma-high tumor versus patients with a stroma-low tumor. On the basis of these findings, a modification in current treatment guidelines will be suggested., Trial Registration: Netherlands Trial Register NTR7270; https://www.trialregister.nl/trial/7072., International Registered Report Identifier (irrid): DERR1-10.2196/13464., (©Marloes Smit, Gabi van Pelt, Annet Roodvoets, Elma Meershoek-Klein Kranenbarg, Hein Putter, Rob Tollenaar, J Han van Krieken, Wilma Mesker. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 14.06.2019.)

Programmed cell death protein 1/programmed cell death protein ligand1 (PD-1/PD-L1) interaction is an important immune checkpoint targeted by anti-PD-1/PD-L1 immunotherapies. However, the observed prognostic significance of PD-1/PD-L1 expression in diffuse large B-cell lymphoma treated with the standard of care has been inconsistent and even contradictory. To clarify the prognostic role of PD-1/PD-L1 expression and interaction in diffuse large B-cell lymphoma, in this study we used 3-marker fluorescent multiplex immunohistochemistry and Automated Quantitative Analysis Technology to assess the CD3 + , PD-L1 + , and PD-1 + CD3 + expression in diagnostic samples and PD-1/PD-L1 interaction as indicated by presence of PD-1 + CD3 + cells in the vicinity of PD-L1 + cells, analyzed their prognostic effects in 414 patients with de novo diffuse large B-cell lymphoma, and examined whether PD-1/PD-L1 interaction is required for the prognostic role of PD-1 + /PD-L1 + expression. We found that low T-cell tissue cellularity, tissue PD-L1 + expression (irrespective of cell types), PD-1 + CD3 + expression, and PD-1/PD-L1 interaction showed hierarchical adverse prognostic effects in the study cohort. PD-1/PD-L1 interaction showed higher sensitivity and specificity than PD-1 + and PD-L1 + expression in predicting inferior prognosis in patients with high CD3 + tissue cellularity ("hot"/inflammatory tumors). However, both PD-1 + and PD-L1 + expression showed adverse prognostic effects independent of PD-1/PD-L1 interaction, and PD-1/PD-L1 interaction showed favorable prognostic effect in PD-L1 + patients without high CD3 + tissue cellularity. Macrophage function and tumor-cell MYC expression may contribute to the PD-1-independent adverse prognostic effect of PD-L1 + expression. In summary, low T-cell tissue cellularity has unfavorable prognostic impact in diffuse large B-cell lymphoma, and tissue PD-L1 + expression and T-cell-derived PD-1 + expression have significant adverse impact only in patients with high T-cell infiltration. PD-1/PD-L1 interaction in tissue is essential but not always responsible for the inhibitory effect of PD-L1 + /PD-1 + expression. These results suggest the benefit of PD-1/PD-L1 blockade therapies only in patients with sufficient T-cell infiltration, and the potential of immunofluorescent assays and Automated Quantitative Analysis in the clinical assessment of PD-1/PD-L1 expression and interaction.

The classification of neuroendocrine neoplasms (NENs) differs between organ systems and currently causes considerable confusion. A uniform classification framework for NENs at any anatomical location may reduce inconsistencies and contradictions among the various systems currently in use. The classification suggested here is intended to allow pathologists and clinicians to manage their patients with NENs consistently, while acknowledging organ-specific differences in classification criteria, tumor biology, and prognostic factors. The classification suggested is based on a consensus conference held at the International Agency for Research on Cancer (IARC) in November 2017 and subsequent discussion with additional experts. The key feature of the new classification is a distinction between differentiated neuroendocrine tumors (NETs), also designated carcinoid tumors in some systems, and poorly differentiated NECs, as they both share common expression of neuroendocrine markers. This dichotomous morphological subdivision into NETs and NECs is supported by genetic evidence at specific anatomic sites as well as clinical, epidemiologic, histologic, and prognostic differences. In many organ systems, NETs are graded as G1, G2, or G3 based on mitotic count and/or Ki-67 labeling index, and/or the presence of necrosis; NECs are considered high grade by definition. We believe this conceptual approach can form the basis for the next generation of NEN classifications and will allow more consistent taxonomy to understand how neoplasms from different organ systems inter-relate clinically and genetically.

Background: In approximately 10% of all gastric cancer (GC) cases, a heritable cause is suspected. A subset of these cases have a causative germline CDH1 mutation; however, in most cases the cause remains unknown. Our objective was to assess to what extent these remaining cases may be explained by germline mutations in the novel candidate GC predisposing genes CTNNA1 , MAP3K6 or MYD88 ., Methods: We sequenced a large cohort of unexplained young and/or familial patients with GC (n=286) without a CDH1 germline mutation for germline variants affecting CTNNA1 , MAP3K6 and MYD88 using a targeted next-generation sequencing approach based on single-molecule molecular inversion probes., Results: Predicted deleterious germline variants were not encountered in MYD88 , but recurrently observed in CTNNA1 (n=2) and MAP3K6 (n=3) in our cohort of patients with GC. In contrast to deleterious variants in CTNNA1 , deleterious variants in MAP3K6 also occur frequently in the general population., Conclusions: Based on our results MAP3K6 should no longer be considered a GC predisposition gene, whereas deleterious CTNNA1 variants are confirmed as an infrequent cause of GC susceptibility. Biallelic MYD88 germline mutations are at most a very rare cause of GC susceptibility as no additional cases were identified., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Neoplastic cell content determination is crucial for biomarker testing. It is known that interobserver variation exists, but largescale data are missing about variation in tumor delineation and cell content determination. Results were obtained from the external quality assessment program for metastatic colorectal cancer from the European Society of Pathology (N = 5776 observations). The study included three parts: current practices were surveyed, neoplastic cell content estimations and delineations were retrieved from stained slides, and clinical reports were analyzed. Seventeen of 43 pathologists determined the neoplastic cell content in a tumor-rich area for DNA extraction and took immune cells (n = 37), tumor cell distribution (n = 33), desmoplastic stroma (n = 30), necrosis (n = 29), and mucus (n = 23) into account. The selected area was highly variable, and the average difference between the highest and lowest estimation ranged between 51% and 78% (2011 to 2017). The number of overestimations was alarmingly high in samples containing <30% tumor cells. Of concern is that 33 of 105 laboratories reported a wild-type result in a sample without tumor in 2017. Standardization of neoplastic cell content determination is needed for test outcome interpretation. The authors' data show variation in estimation practices, tumor delineations and estimations, and interpretation problems (n = 226 reports). Further training for selecting the most suitable block and creating clear reports is urgently needed., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

PTEN loss has been associated with poorer prognosis in many solid tumors. However, such investigation in lymphomas is limited. In this study, PTEN cytoplasmic and nuclear expression, PTEN gene deletion, and PTEN mutations were evaluated in two independent cohorts of diffuse large B-cell lymphoma (DLBCL). Cytoplasmic PTEN expression was found in approximately 67% of total 747 DLBCL cases, more frequently in the activated B-cell-like subtype. Nuclear PTEN expression was less frequent and at lower levels, which significantly correlated with higher PTEN mRNA expression. Remarkably, loss of PTEN protein expression was associated with poorer survival only in DLBCL with AKT hyperactivation. In contrast, high PTEN expression was associated with Myc expression and poorer survival in cases without abnormal AKT activation. Genetic and epigenetic mechanisms for loss of PTEN expression were investigated. PTEN deletions (mostly heterozygous) were detected in 11.3% of DLBCL, and showed opposite prognostic effects in patients with AKT hyperactivation and in MYC rearranged DLBCL patients. PTEN mutations, detected in 10.6% of patients, were associated with upregulation of genes involved in central nervous system function, metabolism, and AKT/mTOR signaling regulation. Loss of PTEN cytoplasmic expression was also associated with TP53 mutations, higher PTEN-targeting microRNA expression, and lower PD-L1 expression. Remarkably, low PTEN mRNA expression was associated with down-regulation of a group of genes involved in immune responses and B-cell development/differentiation, and poorer survival in DLBCL independent of AKT activation. Collectively, multi-levels of PTEN abnormalities and dysregulation may play important roles in PTEN expression and loss, and that loss of PTEN tumor-suppressor function contributes to the poor survival of DLBCL patients with AKT hyperactivation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)